Histone Demethylase KDM5B As a Therapeutic Target for Cancer Therapy

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Review Histone Demethylase KDM5B as a Therapeutic Target for Cancer Therapy

Anmi Jose 1, Gautham G. Shenoy 2, Gabriel Sunil Rodrigues 3, Naveena A. N. Kumar 4 , Murali Munisamy 1, Levin Thomas 1, Jill Kolesar 5 , Ganesha Rai 6, Praveen P. N. Rao 7 and Mahadev Rao 1,*

1 Department of Pharmacy Practice, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; [email protected] (A.J.); [email protected] (M.M.); [email protected] (L.T.) 2 Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; [email protected] 3 Department of Surgery, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; [email protected] 4 Department of Surgical Oncology, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; [email protected] 5 Department of Pharmacy Practice & Science, 567 TODD Building, 789 South Limestone Street, Lexington, KY 40539-0596, USA; [email protected] 6 National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892, USA; [email protected] 7 School of Pharmacy, Health Sciences Campus, 200 University Avenue West, University of Waterloo, Waterloo, ON N2L 3G1, Canada; [email protected] * Correspondence: [email protected]; Tel.: +91-820-2922454

Received: 26 June 2020; Accepted: 26 July 2020; Published: 31 July 2020

Abstract: Lysine-specific demethylase 5B (KDM5B/PLU1/JARID1B) is found to be overexpressed in numerous malignancies, including breast, lung, skin, liver, and prostate cancer. Identification of molecules targeting the KDM5B enzyme could be a potential lead in cancer research. Although many KDM5B inhibitors with promising outcomes have been developed so far, its further application in clinical practice is limited due to toxicity and lack of target specificity. Here, we summarize the significance of targeting KDM5B in anticancer therapy and report the molecular docking studies of some known anti-viral agents, decitabine, entecavir, abacavir, penciclovir, and 3-deazaneplanocin A in the catalytic domain JmjC of KDM5B. These studies show the repurposing potential of identified anti-viral agents in cancer therapy.

Keywords: cancer; histone modiﬁcation; inhibitor; KDM5B; molecular docking; repurposing

1. Introduction Studies over the past few decades and ongoing research reveal that, along with genetic alterations, epigenetic aberrations play a vital part in the evolution and progress of numerous diseases, including several types of cancers [1]. Epigenetic changes, including DNA methylation and histone modiﬁcations, cause altered gene expression without changing the DNA sequence [2,3]. Histone, being the functional protein, not only actively takes part in gene expression, but also acts as a spool for DNA to wind around. Any modiﬁcation on histone molecules via methylation, acetylation, phosphorylation, ubiquitylation, or sumoylation alters the compact chromatin architecture, leading to abnormal gene expression [4]. Histone methylation on arginine or lysine, which are

Cancers 2020, 12, 2121; doi:10.3390/cancers12082121 www.mdpi.com/journal/cancers CancersCancers 20202020,, 1212,, 2121 22 of 1615

which are abundantly present on N- and C- terminal tails of histones, can either turn on or off abundantlytranscription present based onon N-the and degree C- terminal and location tails of of histones, methylation caneither [5]. turn on or off transcription based on theLysine degree-specific and location demethylation of methylation is finely [5]. tuned by two enzyme categories, called lysine-specific demethylaseLysine-specific (KDM1/LSD1) demethylation and the is JumonjiC finely tuned (JmjC) by twodomain, enzyme encompassing categories, histone calledlysine-specific demethylases demethylase(KDMs). JmjC (KDM1, being/LSD1) the largest and the among JumonjiC two (JmjC) families, domain, displays encompassing high substrate histone specificity, demethylases with (KDMs).demethylase JmjC, activity being the targeting largest amonghistones two H3 families, and H4 displays [6]. In mammals, high substrate around specificity, thirty KDMs with demethylase have been activityidentified targeting and grouped histones asH3 phylogenetically and H4 [6]. In mammals, distinct aroundsubfamilies thirty from KDMs KDM2 have been-8 with identified numerous and groupedsubcategories as phylogenetically [7]. distinct subfamilies from KDM2-8 with numerous subcategories [7]. The KDM5KDM5/JARID1/JARID1 subfamily subfamily consists consists of four of interrelatedfour interrelated histone histone demethylase demethylase enzymes—KDM5A, enzymes— KDM5B,KDM5A, KDM5CKDM5B, and KDM5C KDM5D—involved and KDM5D— ininvolved several in human several biological human biological processes, processes including, hormonalincluding response,hormonal stem response, cell regeneration, stem cell genomic regeneration, stability, genomic cellular stability, proliferation, cellular and di proliferation,fferentiation [8 and,9]. KDM5Bdifferentiation (also known [8,9]. KDM5B as PLU1 (also or JARID1B), known as demethylates PLU1 or JARID1B lysine),4 demethylate of histone 3s (H3K4), lysine 4 and of histone acts as a3 transcriptional(H3K4), and acts repressor as a transcriptional on certain tumor repressor suppressor on certain genes, tumor thus converting suppressor it genes, into the thus transcriptionally converting it inactiveinto the statetranscriptionally [10]. The deregulated inactive expression state [10]. ofThe KDM5B deregulated has been expression implicated of in KDM5B numerous has cancer been types,implicated including in numerous breast, lung, cancer skin, types, prostate, including testis, and breast, liver lung, [11]. skin, Figure prostate,1 depicts testis, the top and ten liver cancers, [11]. withFigure high 1 depicts KDM5B th mRNAe top ten expression cancers, data with extracted high KDM5B from cBioPortal mRNA expression for cancer data genomics—TCGA, extracted from PanCancercBioPortal forAtlas cancer study g [enomics12–14].— Thus,TCGA, KDM5B PanCancer could be Atlas considered study [12 as– a14 potential]. Thus, therapeutic KDM5B could target be inconsidered precision as oncology. a potential Hence, therapeutic drugs target that canin precision selectively oncology. inhibit Hence KDM5B, drugs could that be can of substantial selectively importanceinhibit KDM5B as a could therapeutic be of substantial intervention importance for such cancer as a therapeutic types. This intervention review attempts for such to comprehend,cancer types. asThis thoroughly review attempts as possible, to thecomprehend oncogenic, roleas thoroughly of KDM5B inas majorpossible, cancer the types oncogenic and the role drug of repurposing KDM5B in potentialmajor cancer of some types known and the small drug molecules repurposing by targeting potential KDM5B’s of some known catalytic small domain molecules through by molecular targeting dockingKDM5B’s studies. catalytic domain through molecular docking studies.

Figure 1. PictorialPictorial representation representation of ofthe the top top ten tencancers cancers with with highhigh KDM5B KDM5B mRNA mRNA expression expression.. Data Dataextracted extracted from the from cBioPortal the cBioPortal for cancer for genomics cancer genomics—TCGA,—TCGA, PanCancer PanCancer Atlas (n = n Atlasumber (n of= patientsnumber). of patients). 2. Structure and Enzymatic Function of KDM5B The JmjC family is reliant on Fe (II) and α- ketoglutarate co-factors for its demethylating activity. KDM5B gene consists of 4632 bases, positioned on chromosome 1, in the cytogenetic band of 1q32.1 with a molecular mass of 175 kilodaltons. It encodes a protein of 1544 amino acids [15] comprising an N-terminal Jumonji (JmjN) domain, a catalytic JmjC domain, an AT-rich domain (ARID), a C5HC2

Cancers 2020, 12, 2121 3 of 16

2. Structure and Enzymatic Function of KDM5B The JmjC family is reliant on Fe (II) and α- ketoglutarate co-factors for its demethylating activity. KDM5B gene consists of 4632 bases, positioned on chromosome 1, in the cytogenetic band of 1q32.1 with a molecular mass of 175 kilodaltons. It encodes a protein of 1544 amino acids [15] comprising an N-terminal Jumonji (JmjN) domain, a catalytic JmjC domain, an AT-rich domain (ARID), a C5HC2 zinc finger domain, a PLU1 motif and three plant homeobox domain (PHD1, PHD2, and PHD3). The domain architecture of KDM5B is highly conserved and homologous from yeast to humans. JmjN and JmjC are vital domains required for enzymatic activity, whereas AT-rich domain binds to GC rich DNA sequences. PHD domains contribute to substrate recognition in which it prevents reverse reaction [16–21]. ARID and PHD domains have very less influence, whereas JmjN and JmjC along with C5HC2 zinc finger domain have more impact on the KDM5B’s catalytic activity. The overall catalytic core is composed of three conserved domains consisting of: (i) the JmjN, JmjC, and ARID domains (residues 31–72, 375–602 and 94–100, respectively); (ii) a C-terminal helical domain (residues 604–671 and 737–753); (iii) a β-sheet composed of three β-strands (residues 673–734) that harbored a C5HC2 zinc finger motif [20].

3. Signiﬁcance of KDM5B in Various Cancers

3.1. Breast Cancer Although the consistent expression of KDM5B in breast cancer was established by Lu P.J. et al. [22] in 1999, Yamane K. et al. in 2007 revealed the salient role of KDM5B in breast cancer cell proliferation via the transcriptional repression of tumor suppressor genes, including BRCA1 [19]. Subsequent studies both in vitro and in vivo, involving gene expression and KDM5B knockdown, confirmed its putative role in breast tumorigenesis [19,23–25]. One of the proposed mechanisms of KDM5B-mediated tumor cell proliferation was by repressing tumor suppressor miRNA let-7e [26]. Besides, the TFAP2C-Myc-KDM5B complex can repress p21, leading to tumorigenesis and therapy failure [27]. A recent study showed that estrogen receptor-positive (ER+) tumors, with KDM5B overexpression, had poor clinical outcomes and resistance to hormonal therapy [25]. Moreover, KDM5B expression was found to be negatively correlated with p16 protein expression [28]. Significantly, microRNA hsa-miR-448 can suppress KDM5B expression through MALAT1 and can prevent triple-negative breast cancer (TNBC) progression [29]. The downregulation of KDM5B led to 30UTR lengthening of the cyclin D1 (CCND1) oncogene and lengthening of a tumor suppressor gene, DICER1, suggesting KDM5B as a novel target for 30UTR processing [30]. KDM5B inhibition promoted the re-expression of tumor suppressor protein HEXIM1, and upregulated HEXIM1 aided in the inhibition of breast cancer cell proliferation using KDM5B inhibitors [31]. Recently, Paroni G. et al. showed that HER2–positive breast cancer cells were sensitive to KDM5 inhibition and KDM5 inhibitors exhibited a synergistic effect with HER2 targeting drugs, trastuzumab and lapatinib [32]. Similarly, numerous studies have reported the oncogenic role of KDM5B, where gene expression levels were related with poor prognosis, cancer cell proliferation, and metastasis [33–35]. Figure2 shows the KDM5B mRNA overexpression in breast cancer from various studies retrieved from the cBioPortal for cancer genomics [12–14,36–38]. Cancers 2020, 12, 3 of 15 zinc finger domain, a PLU1 motif and three plant homeobox domain (PHD1, PHD2, and PHD3). The domain architecture of KDM5B is highly conserved and homologous from yeast to humans. JmjN and JmjC are vital domains required for enzymatic activity, whereas AT-rich domain binds to GC rich DNA sequences. PHD domains contribute to substrate recognition in which it prevents reverse reaction [16–21]. ARID and PHD domains have very less influence, whereas JmjN and JmjC along with C5HC2 zinc finger domain have more impact on the KDM5B’s catalytic activity. The overall catalytic core is composed of three conserved domains consisting of: (i) the JmjN, JmjC, and ARID domains (residues 31–72, 375–602 and 94–100, respectively); (ii) a C-terminal helical domain (residues 604–671 and 737–753); (iii) a β-sheet composed of three β-strands (residues 673–734) that harbored a C5HC2 zinc finger motif [20].

3. Significance of KDM5B in Various Cancers

FigureFigure 2. 2. AA graphical graphical representation representation of of the the high high KDM5B KDM5B mRNA mRNA expression expression among among breast breast cancer cancer patientspatients.. D Dataata extracted extracted from from the the cBioPortal cBioPortal for for cancer cancer genomics genomics.. 3.2. Lung Cancer KDM5B expression rate was found to be highly elevated in neoplastic tissues, in contrast to normal tissues, irrespective of lung carcinoma histology [39–41]. The suppression of KDM5B expression showed a signiﬁcant reduction in cancer cell growth via the E2F/RB1 pathway [40]. Han L. et al. reported in 2013 that KDM5B positively regulates brain metastasis in NSCLC [42]. Moreover, KDM5B aids in the proliferation, invasion, and metastasis activities of lung cancer cells through downregulated p53 [39]. Besides, Shen X. et al. reported that KDM5B overexpression positively correlated with size and stage of the tumor, lymph node metastasis, and reduced survival rate [39]. A recent study by Lu Y. et al. established that KDM5B plays crucial roles in hypoxia-induced geﬁtinib (EGFR TKI) resistance and epithelial–mesenchymal transition (EMT) [43].

3.3. Melanoma The role of KDM5B in melanoma is still controversial. Roesch A. et al., in their studies, showed that KDM5B promoted tumor cell maintenance and metastasis, and its knockdown led to the exhaustion of melanoma cells [44]. However, KDM5B has also been suggested as a tumor-suppressive agent in melanocytic cells through the regulation of activities of the retinoblastoma protein [45,46]. Interestingly, it is evident that, in melanoma cells, KDM5B expression is dynamically regulated. However, KDM5B was overexpressed in certain benign tumors, whereas downregulated in aggressive and metastatic melanomas [47]. The relevance of KDM5B in chemo-resistance was conﬁrmed when KDM5B depletion led to the increased sensitivity of melanocytes to anti-melanoma treatment [48].

3.4. Hepatocellular Carcinoma KDM5B was often upregulated in hepatocellular carcinoma (HCC) samples, compared to corresponding non-neoplastic tissues. Besides, the gene expression level was associated with tumor properties and stages of cancer. HCC patients with KDM5B overexpression had more tumor metastasis, poor prognosis, and signiﬁcantly shorter overall survival [49–52]. KDM5B exerts its oncogenic function by inactivating PTEN transcription [49] and by silencing KDM5B, which, remarkably, hinders cancer cell proliferation through p15 and p27 up-regulation [50].

3.5. Gastric Cancer The aberrant expression of KDM5B mRNA was detected in gastric tumor specimens and cell lines in comparison with normal tissues. The expression rate also correlated with TNM stage and depth of invasion [53]. Various studies conﬁrm the contribution of KDM5B in various signaling pathways for tumor cell development and migration [53,54]. Bao J. et al. reported that miR-194, Cancers 2020, 12, 2121 5 of 16 which obstructs gastric cancer cell growth and invasion, directly targets and negatively regulates KDM5B [55]. These ﬁndings revealed the potential role of KDM5B, as a tumor promoter in gastric cancer. A recent study by Xu W. et al. described that increased KDM5B expression, associated with cisplatin resistance in gastric cancer cell lines and targeting KDM5B, reversed the phenotype [56].

3.6. Colorectal Cancer The functional signiﬁcance of KDM5B in colorectal cancer (CRC) stem cells was conﬁrmed through in vitro and in vivo studies. KDM5B knockdown was found to surge H3K4 trimethylation at the p16/INK4A tumor suppressor’s promoter region, which subsequently induced cellular senescence [57]. Besides, KDM5B was found to be overexpressed in CRC tumor tissue compared with normal colon samples and this overexpression positively correlated with cancer progression [58].

3.7. Bladder Cancer Knockdown and apoptosis studies demonstrated the close link of KDM5B, in cell cycle regulation and cancer cell maintenance, in bladder cancer cell lines. Overexpression of KDM5B was noted in the early and advanced stages of bladder cancer, irrespective of cancer grade [40]. A potential inverse connection between KDM5B and connexin 26 (CX26) in the progression of bladder cancer was reported, where KDM5B was found to be upregulated in contrast to CX26 [59].

3.8. Prostate Cancer KDM5B was found to be overexpressed in prostate cancer tissues, compared to benign tissues, and has been suggested as a potential therapeutic target [15,60]. Various studies evaluated the role of microRNAs in regulating KDM5B. The forced miR-29a expression inhibited proliferation, and induced apoptosis in prostate cancer cells, by repressing KDM5B expression, whereas, by KDM5B targeting, miR-137 aided as a tumor suppressor in prostate carcinogenesis [61,62].

3.9. KDM5B in Other Cancers KDM5B showed signiﬁcantly high expression levels in patients with B-cell precursor acute lymphoblastic leukemia (B-ALL), compared to normal bone marrow [63]. The suppression of KDM5B hindered metastasis and the invasion of human oral squamous cell carcinoma and sensitized radiation therapy [64]. KDM5B overexpression was signiﬁcantly associated with tumor cell proliferation in head and neck cancer, and KDM5B silencing caused cell growth suppression both in vitro and in vivo [65]. Similar studies supporting the oncogenic role of KDM5B were observed in other malignancies, such as esophageal squamous cell carcinoma [66], cervical cancer [67], renal cell carcinoma [68], epithelial ovarian cancer [69] and neuroblastoma [70].

3.10. KDM5B as a Therapeutic Target Apart from the oncogenic and tumorigenic role, KDM5B is also considered as a cancer-testis antigen (CTA), where gene expression is restricted in normal tissues except adult testes [22,71]. Therefore, abnormal overexpression of KDM5B in any neoplastic tissue, in comparison with corresponding non-neoplastic tissues, makes it an ideal therapeutic target. Furthermore, CTA properties suggest that targeting KDM5B may be associated with reduced toxicity. Therefore, the development of specific KDM5B inhibitors will offer a deeper insight into the therapeutic potential for many types of cancers. As Fe (II) and α- ketoglutarate are the two vital co-factors of KDM5B in demethylation, studies are focusing on the development of compounds that mimic α-ketoglutarate or compounds that chelate with Fe (II). Over the years, many KDM5B inhibitors have been developed (Figure3) and have provided promising outcomes [72]. The 2,4-pyridinedicarboxylic acid (2,4-PDCA) inhibits KDM5B in vitro with an IC value of 3 1 µM[5], whereas GSK-J1 with an IC of 0.55 µM[20,73]. GSK467 displayed an 50 ± 50 inhibitory concentration of 0.026 µM. The crystalline structure of KDM5B with GSK467 provides a Cancers 2020, 12, 2121 6 of 16 possible template for the development of selective KDM5B inhibitors [20,74]. Compounds 54j and 54k are potent, cell permeable dual inhibitors of the KDM4 and KDM5 subfamilies. Compounds 54j and 54k inhibit KDM5B with IC50 values 0.014 µM and 0.023 µM, respectively, and have shown adequate cellular permeability [75]. CPI-455 with IC50 of 0.003 µM, was identified as a pan-KDM5 inhibitor that competitively binds to the two cofactor binding sites [76]. KDM5-C49, a 2,4-PDCA analogue, and further modified KDOAM-25 have shown acceptable inhibitory potency on KDM5B, in which KDOAM-25 exhibited good stability, high selectivity and low cytotoxicity [20,77]. An orally available, potent inhibitor of KDM5B, named compound 33, was identified through structure activity relationship exploration and presented promising selectivity compared to early inhibitors [78]. Recently, a pyrazole derivative, compound 27ab, was discovered, which inhibited MKN45 cell proliferation, wound healing and migration [79]. All these inhibitors are in their initial stage of development and have not made it toCanc theers clinics2020, 12,yet. 6 of 15

Figure 3. Previously reported KDM5B inhibitors (IC50: half maximal inhibitory concentration). Figure 3. Previously reported KDM5B inhibitors (IC50: half maximal inhibitory concentration). 4. Molecular Docking Studies 4. Molecular Docking Studies The JmjC domain of native KDM5B, which is involved in histone demethylation, contains Fe (II) ion and αThe-ketoglutarate JmjC domain as of cofactors. native KDM5B, This site which is an attractive is involved target in histone to develop demethylation, KDM5B inhibitors. contains One Fe (II) of theion solvedand α- crystalketoglutarate structures as cofactors. of KDM5B, This with site an is inhibitor an attractive GSK467 target in the to JmjC develop domain, KDM5B contains inhibitors. Mn (II) insteadOne of ofthe Fe solved (II) [20 crystal]. This structuresstructure provides of KDM5B, an excellent with an starting inhibitor point GSK467 to develop in the novel JmjC domain,KDM5B inhibitorscontains Mn using (II) computationalinstead of Fe (II) tools. [20]. Accordingly,This structure we pr usedovides computational an excellent starting modeling point to exploreto develop the potentialnovel KDM5B of known inhibitors drugs using to inhibit computational KDM5B. Drug tools. repurposing Accordingly, is an we approach used computational where new therapeutic modeling indicationsto explore the for potential already marketed of known drugs drugs are to discovered. inhibit KDM5B. This approach Drug repurposing aims to lessen is an the approach price and where time involvednew therapeutic in drug indi discoverycations process for already [80,81 marketed]. On this drugs note, weare considered discovered. the This drug approach repurposing aims potentialto lessen ofthe nucleoside price and time derivatives, involved which in drug are discovery well known process as antiviral [80,81] agents. On this [82 note,]. Several we considered studies in the the drug past haverepurposing shown that potential nucleoside of nucleoside derivatives, derivatives, with monocyclic which or are bicyclic well known ring systems, as antiviral exhibit agents anticancer [82]. propertiesSeveral studies [83– 85in]. the We past investigated have shown the that drug nucleoside repurposing derivatives, potential with of known monocyclic compounds, or bicyclic such ring as decitabine,systems, exhibit entecavir, anticancer abacavir, properties penciclovir, [83– and85]. 3-deazaneplanocin We investigated the A drug (DZNep) repurposing (Figure4 ), potential which are of allknown nucleoside compounds derivatives,, such as as decitabine, inhibitors ofentecavir, KDM5B, abacavir, by conducting penciclovir molecular, and docking3-deazaneplanocin experiments. A In(DZNep) addition, (Figure we compared 4), which the are binding all nucleoside modes ofderivatives, previously as reported inhibitors KDM5B of KDM5B inhibitors, by conducting containing planarmolecular bicyclic docking rings, experiments. such as GSK467, In addition, 54j, and we CPI-455, compared with the penciclovirbinding modes and of DZNep, previously to determine reported KDM5B inhibitors containing planar bicyclic rings, such as GSK467, 54j, and CPI-455, with penciclovir and DZNep, to determine their interactions in the JmjC domain of KDM5B. It should be noted that decitabine (Dacogen®) is currently used to treat myelodysplastic syndrome (MDS), entecavir (Baraclude®) is used as an anti-hepatitis B agent, abacavir (Ziagen®) is used to treat HIV infections and penciclovir (Denavir®) is a known drug used in the treatment of herpes infections. The carbocyclic adenosine derivative DZNep, is a known inhibitor of histone methyltransferase EZH2 and is a promising compound in cancer immunotherapy [86]. Molecular docking studies were carried out using the computational software, Discovery Studio (DS) Structure-Based-Design (BIOVIA Inc, Dassault Systemes, USA). Moreover, previous studies have shown that nucleoside derivatives with either a monocyclic or bicyclic ring exhibit anticancer property. The ligands decitabine, entecavir, abacavir, penciclovir, and DZNep were built in 3D, using the Small Molecules module in DS using CHARMm force field. The X-ray crystal structure of human KDM5B was obtained from PDB (pdb id: 5FUN) [20]. This KDM5B structure contains a pyridopyrimidine ligand GSK467 and the catalytic site contains Mn2+ ion. All the water molecules were removed and a 10 Å binding site sphere was defined by selecting the bound ligand GSK467, after which it was deleted. The KDM5B protein was prepared

Cancers 2020, 12, 2121 7 of 16 their interactions in the JmjC domain of KDM5B. It should be noted that decitabine (Dacogen®) is currently used to treat myelodysplastic syndrome (MDS), entecavir (Baraclude®) is used as an anti-hepatitis B agent, abacavir (Ziagen®) is used to treat HIV infections and penciclovir (Denavir®) is a known drug used in the treatment of herpes infections. The carbocyclic adenosine derivative DZNep, is a known inhibitor of histone methyltransferase EZH2 and is a promising compound in cancer immunotherapy [86]. Molecular docking studies were carried out using the computational software, Discovery Studio (DS) Structure-Based-Design (BIOVIA Inc, Dassault Systemes, USA). Moreover, previous studies have shown that nucleoside derivatives with either a monocyclic or bicyclic ring exhibit anticancer property. The ligands decitabine, entecavir, abacavir, penciclovir, and DZNep were built in 3D, using the Small Molecules module in DS using CHARMm force field. The X-ray crystal structure of human KDM5B was obtained from PDB (pdb id: 5FUN) [20]. This KDM5B structure contains a pyridopyrimidine ligand GSK467 and the catalytic site contains Mn2+ ion. All the water molecules were Cancers 2020, 12, 7 of 15 removed and a 10 Å binding site sphere was defined by selecting the bound ligand GSK467, after which itusing was the deleted. Macromolecules The KDM5B module protein in wasDS using prepared CHARMm using theforce Macromolecules field. Docking was module carried in DSout usingusing CHARMmthe LibDock force algorithm field. Docking by employing was carried 100 out hotspots, using the a LibDockdocking algorithmtolerance byof employing0.25 Å, and 100 an hotspots, implicit asolvent docking model, tolerance with ofa distance 0.25 Å,- anddependent an implicit dielectric solvent constant model, and with CHARMm a distance-dependent force field. The dielectric binding constantposes obtained, and CHARMm were further force field. energy The minimized binding poses using obtained, Smart wereminimizer further (1000 energy steps minimized and an using RMS Smartgradient minimizer of 0.001 (1000 kcal/mol) steps and an ranked RMS usinggradient the of LibDock 0.001 kcal scoring/mol) and function. ranked The using ligand the LibDock-binding scoringinteractions function. were The further ligand-binding studied by interactions considering were the polarfurther and studied nonpolar by considering contacts in the the polar KDM5B and nonpolarbinding site contacts. The in general the KDM5B computational binding site. methods The general applied computational were as per methods our previously applied reported were as permethod ourpreviously [87]. reported method [87].

FigureFigure 4.4. Selected antiviral drugs targeting KDM5B.KDM5B.

DockingDocking the the nucleoside nucleoside decitabine decitabine in the in KDM5B the KDM5B catalytic catalytic site shows site that shows the 6-member that the azacytidine 6-member ringazacytidine was in theringα -ketoglutaratewas in the α-ketoglutarate binding pocket binding and underwentpocket and πunderwent–π stacked π hydrophobic–π stacked hydrophobic interactions withinteractions aromatic with rings aromatic of Tyr488 rings and of Tyr488 Phe496, and respectively Phe496, respectively (distance < (distance4.1 Å). The < 4.1 azacytidine Å). The azacytidine N3 was 2+ not forming any metal-ligand bonding with Mn center2+ (distance 5.0 Å, Figure5, Panel A). N3 was not forming any metal-ligand bonding with Mn center (distance≈ ≈ 5.0 Å, Figure 5, Panel A). TheThe deoxydeoxy sugarsugar moietymoiety waswas inin aa regionregion comprisedcomprised ofof Tyr425,Tyr425, Gly426,Gly426, andand Ser495.Ser495. TheThe hydroxyhydroxy andand the hydroxymethyl groups were in contact with Ser495 and Gly426 backbones, via hydrogen bonding interaction (distance < 1.9 Å, Figure 5, Panel A). These studies suggest that decitabine can undergo favorable interactions with KDM5B. Docking entecavir in KDM5B shows that the bicyclic guanosine ring was oriented in the α- ketoglutarate binding pocket, where it underwent some favorable interactions. The pyrimidine and imidazole rings were in contact with Tyr488 and Phe496 via four π–π stacked hydrophobic interactions (distance < 4.5 Å, Figure 5, Panel B). Interestingly, the guanosine C6 ketone (C = O) underwent metal–ligand bonding with the Mn2+ center (distance = 2.6 Å). The exocyclic alkene was in contact with Tyr425, Ala427, and Tyr488 (distance < 5.0 Å), whereas the hydroxy group of the cyclopentenyl ring formed a hydrogen bond with the backbone of Ser495 (distance < 2.0 Å, Figure 5, Panel B). In general, entecavir exhibited better binding interactions with KDM5B compared to decitabine. This can be attributed to the presence of a planar bicyclic guanosine ring in entecavir, and the metal–ligand bond with the guanosine ketone.

Cancers 2020, 12, 2121 8 of 16 the hydroxymethyl groups were in contact with Ser495 and Gly426 backbones, via hydrogen bonding interaction (distance < 1.9 Å, Figure5, Panel A). These studies suggest that decitabine can undergo

Cancfavorableers 2020 interactions, 12, with KDM5B. 8 of 15

Figure 5. The binding modes of decitabine (A), entecavir (B), abacavir (C) and penciclovir (D) in ball Figure 5. The binding modes of decitabine (A), entecavir (B), abacavir (C) and penciclovir (D) in ball and stick cartoon within the JmjC domain of human KDM5B (pdb id: 5FUN). Mn2+ ion is shown and stick cartoon within the JmjC domain of human KDM5B (pdb id: 5FUN). Mn2+ ion is shown as a as a green circle. Hydrogen atoms are removed to enhance clarity. Polar and nonpolar interactions green circle. Hydrogen atoms are removed to enhance clarity. Polar and nonpolar interactions are are color-coded. color-coded. Docking entecavir in KDM5B shows that the bicyclic guanosine ring was oriented in the Docking the anti-HIV agent abacavir with KDM5B shows that it was buried in the α- α-ketoglutarate binding pocket, where it underwent some favorable interactions. The pyrimidine and ketoglutarate binding pocket closer to the metal center similar to entecavir, and the purine ring imidazole rings were in contact with Tyr488 and Phe496 via four π–π stacked hydrophobic interactions underwent π–π stacking interactions with aromatic rings of Tyr488 and Phe496 (distance < 4.8 Å) and (distance < 4.5 Å, Figure5, Panel B). Interestingly, the guanosine C6 ketone (C = O) underwent the N1 of purine ring was in contact with Mn2+ (distance = 2.6 Å). Furthermore, the C2 amino metal–ligand bonding with the Mn2+ center (distance = 2.6 Å). The exocyclic alkene was in contact with substituent underwent hydrogen bonding interaction with the backbone of Asn509 (distance < 1.9 Å, Tyr425, Ala427, and Tyr488 (distance < 5.0 Å), whereas the hydroxy group of the cyclopentenyl ring Figure 5, Panel C). The cyclopentenyl ring was in contact with Tyr488 and Phe496 via hydrophobic formed a hydrogen bond with the backbone of Ser495 (distance < 2.0 Å, Figure5, Panel B). In general, interactions (distance < 4.8 Å). entecavir exhibited better binding interactions with KDM5B compared to decitabine. This can be Docking another purine-based drug penciclovir, which contains a guanosine ring similar to attributed to the presence of a planar bicyclic guanosine ring in entecavir, and the metal–ligand bond entecavir in the KDM5B, shows that it was exhibiting a similar binding mode as entecavir, and the with the guanosine ketone. guanosine ring was in hydrophobic contact with Tyr488 and Phe496 by forming several π–π stacking Docking the anti-HIV agent abacavir with KDM5B shows that it was buried in the α-ketoglutarate interactions (distance < 4.4 Å, Figure 5, Panel D). The guanosine ketone forms a metal–ligand binding pocket closer to the metal center similar to entecavir, and the purine ring underwent π–π interaction with Mn2+ (distance = 2.4 Å). The acyclic sugar moiety was closer to a polar region stacking interactions with aromatic rings of Tyr488 and Phe496 (distance < 4.8 Å) and the N1 of comprised of Gly426, Ser494, Val489, and Asn591. The hydroxy and hydroxymethyl substituents purine ring was in contact with Mn2+ (distance = 2.6 Å). Furthermore, the C2 amino substituent form hydrogen-bonding interactions with Gly429, Ser494, and Val489 backbones (distance < 2.8 Å, underwent hydrogen bonding interaction with the backbone of Asn509 (distance < 1.9 Å, Figure5, Figure 5, Panel D). These studies show that drugs containing a bicyclic purine template exhibit Panel C). The cyclopentenyl ring was in contact with Tyr488 and Phe496 via hydrophobic interactions favorable binding in KDM5B. (distance < 4.8 Å). Docking DZNep, an investigational drug molecule, which also contains a planar bicyclic imidazo[4,5-c]pyridine ring template (Figure 4), and a cyclopentenyl moiety, shows that the bicyclic imidazo[4,5-c]pyridine ring, forms a π–π stacking interaction with Tyr488 and Phe496 (distance < 4.3 Å). Interestingly, the pyridine nitrogen, chelated with Mn2+ (distance = 2.8 Å, Figure 6A) and C2 amino substituents formed a hydrogen bond interaction with backbone of Asn509. The cyclopentenyl hydroxyl and hydroxymethyl substituents also underwent hydrogen bonding interactions with Gly426 and Ser495 (distance < 2.0 Å).

Cancers 2020, 12, 2121 9 of 16

Docking another purine-based drug penciclovir, which contains a guanosine ring similar to entecavir in the KDM5B, shows that it was exhibiting a similar binding mode as entecavir, and the guanosine ring was in hydrophobic contact with Tyr488 and Phe496 by forming several π–π stacking interactions (distance < 4.4 Å, Figure5, Panel D). The guanosine ketone forms a metal–ligand interaction with Mn2+ (distance = 2.4 Å). The acyclic sugar moiety was closer to a polar region comprised of Gly426, Ser494, Val489, and Asn591. The hydroxy and hydroxymethyl substituents form hydrogen-bonding interactions with Gly429, Ser494, and Val489 backbones (distance < 2.8 Å, Figure5, Panel D). These studies show that drugs containing a bicyclic purine template exhibit favorable binding in KDM5B. Docking DZNep, an investigational drug molecule, which also contains a planar bicyclic imidazo[4,5-c]pyridine ring template (Figure4), and a cyclopentenyl moiety, shows that the bicyclic imidazo[4,5-c]pyridine ring, forms a π–π stacking interaction with Tyr488 and Phe496 (distance < 4.3 Å). Interestingly, the pyridine nitrogen, chelated with Mn2+ (distance = 2.8 Å, Figure6A) and C2 amino substituentsCancers 2020, 12, formed a hydrogen bond interaction with backbone of Asn509. The cyclopentenyl9 of 15 hydroxyl and hydroxymethyl substituents also underwent hydrogen bonding interactions with Gly426 and Ser495Among (distance these compounds,< 2.0 Å). DZNep exhibited the most favorable binding interaction with KDM5B.Among The thesebinding compounds, interaction DZNep was measured exhibited by the calculating most favorable the LibDock binding scores interaction for the with best KDM5B.binding Theposes. binding The ranking interaction order was was measured DZNep (LibDock by calculating score = the 131.60) LibDock > penciclovir scores for (LibDock the best bindingscore = 129.84) poses. The> entecavir ranking (LibDock order was score DZNep = 121.26) (LibDock > abacavir score (LibDock= 131.60) score> penciclovir = 118.10) > decitabine (LibDock score(LibDock= 129.84) score >= entecavir 109.55). Molecular (LibDock docking score = studies121.26) > of abacavir known KDM5B (LibDock inhibitor score =, 54j118.10) (Figure> decitabine 6B), shows (LibDock that its scorebicyclic= 109.55).pyridopyrimidine Molecular dockingring underwent studies of van known der Waal’s KDM5B contact inhibitor, with 54j Tyr488 (Figure and6B), Phe496 shows (π that–π itsstacked bicyclic interactions, pyridopyrimidine distance < ring 5 Å underwent) and the pyrimidinone van der Waal’s ketone contact formed with a Tyr488hydrogen and bond Phe496 with (π the–π stackedside chain interactions, of Lys517 distance(distance< =5 Å)1.55 and Å). the Interest pyrimidinoneingly, the ketone nitrogen formed of pyrazole a hydrogen and bondpyrimidinone with the siderings chain was ofclose Lys517 to the (distance metal =center1.55, Å). indicating Interestingly, the ability the nitrogen of 54j ofto pyrazole form metal and– pyrimidinoneligand interactions rings was(Figure close 6B). to theThe metal piperidine center, benzene indicating with the 3,5 ability-dichloro of 54j substituents, to form metal–ligand was oriented interactions in a hydrophobic (Figure6B). Thepocket piperidine made up benzene of Ile500, with Val553 3,5-dichloro and Tyr586. substituents, Furthermore, was oriented 54j is a inmuch a hydrophobic larger molecule pocket (molecular made up ofvolume Ile500, = Val553 345.7 Å and3) compared Tyr586. Furthermore, to GSK467,54j CPI is- a455, much decitabine, larger molecule entecavir, (molecular abacavir, volume penciclovir= 345.7, and Å3) comparedDZNep (molecular to GSK467, volume CPI-455, range decitabine, ~ 168.7–231.5 entecavir, Å3). Therefore abacavir,, 54j penciclovir, is able to bind and in DZNep the entire (molecular span of volumethe JmjC range domain ~ 168.7–231.5 (Figure 6B), Å 3which). Therefore, is not possible 54j is able for to the bind antivirals in the entire investigated span of thein this JmjC study domain, as (Figurethey are6 B),smaller. which This is not is also possible supported for the by antivirals a superior investigated LibDock score in thisobtained study, for as 54j they (LibDock are smaller. score This= 145.08). is also supportedHowever, bysimilar a superior to 54j LibDock, all the score bicyclic obtained ring for-containing 54j (LibDock agents score, entecavir,= 145.08). However, abacavir, similarpenciclovir to 54j,, and all DZNep the bicyclic, are able ring-containing to undergo metal agents,–ligand entecavir, interaction abacavir, (Figures penciclovir, 5 and 6 and), which DZNep, is a arecommon able to feature undergo observed. metal–ligand interaction (Figures5 and6), which is a common feature observed.

Figure 6.6. TheThe bindingbinding mode mode of of DZNep DZNep (A (),A 54j), 54j (B )(B and) and CPI-455 CPI-455 (C) (ball(C) (ball and and stick stick cartoon) cartoon) within within JmjC 2+ domainJmjC domain of human of human KDM5B KDM5B (pdb id:(pdb 5FUN). id: 5FUN). Mn Mnion2+ ision shown is shown as a as green a green circle. circle. Hydrogen Hydrogen atoms atoms are removed to enhance clarity. Polar and nonpolar interactions are color-coded. are removed to enhance clarity. Polar and nonpolar interactions are color-coded.

Molecular docking studies of the known KDM5B inhibitor, CPI-455 (Figure 6C), shows that it was oriented in a similar region compared to molecules containing the planar bicyclic ring investigated in this study. The bicyclic pyrazolopyrimidinone ring of CPI-455 was in van der Waal’s contact with Tyr488 and Phe496 (distance < 5 Å), and the pyrimidinone ketone underwent a hydrogen-bonding interaction with the Lys517 side chain (distance = 2.48 Å). The C6 isopropyl substituent was in a hydrophobic pocket and underwent multiple interactions with Tyr425 and Ala427 (distance < 5 Å), whereas the C5 phenyl ring underwent π–π stacking interactions with Tyr425 (distance < 5 Å). Interestingly, the C3 nitrile substituent was able to chelate with the metal center (distance = 2.6 Å, Figure 6C) which shows the significance of a nitrile substituent in KDM5B binding (LibDock score = 120.19). These molecular docking studies also show that bicyclic ring-containing agents (e.g., 54j, CPI-455, entecavir, abacavir, penciclovir, and DZNep), exhibit better binding in the JmjC domain of KDM5B, compared to monocyclic ring-containing decitabine, which is also reflected in a lower LibDock score (109.55) obtained for decitabine compared to other molecules. The binding modes of DZNep and penciclovir, which exhibited superior LibDock scores, were compared with the known X-ray structure of GSK467 (Figure 3), bound to KDM5B. This investigation shows that, similar to GSK467, the planar bicyclic rings of DZNep and penciclovir were oriented in the α-ketoglutarate binding pocket, and more significantly, the pyridine nitrogen of DZNep and guanosine ketone of penciclovir were in contact with the metal center Mn2+ (Figure 7). These

Cancers 2020, 12, 2121 10 of 16

Molecular docking studies of the known KDM5B inhibitor, CPI-455 (Figure6C), shows that it was oriented in a similar region compared to molecules containing the planar bicyclic ring investigated in this study. The bicyclic pyrazolopyrimidinone ring of CPI-455 was in van der Waal’s contact with Tyr488 and Phe496 (distance < 5 Å), and the pyrimidinone ketone underwent a hydrogen-bonding interaction with the Lys517 side chain (distance = 2.48 Å). The C6 isopropyl substituent was in a hydrophobic pocket and underwent multiple interactions with Tyr425 and Ala427 (distance < 5 Å), whereas the C5 phenyl ring underwent π–π stacking interactions with Tyr425 (distance < 5 Å). Interestingly, the C3 nitrile substituent was able to chelate with the metal center (distance = 2.6 Å, Figure6C) which shows the significance of a nitrile substituent in KDM5B binding (LibDock score = 120.19). These molecular docking studies also show that bicyclic ring-containing agents (e.g., 54j, CPI-455, entecavir, abacavir, penciclovir, and DZNep), exhibit better binding in the JmjC domain of KDM5B, compared to monocyclic ring-containing decitabine, which is also reflected in a lower LibDock score (109.55) obtained for decitabine compared to other molecules. The binding modes of DZNep and penciclovir, which exhibited superior LibDock scores, were compared with the known X-ray structure of GSK467 (Figure3), bound to KDM5B. This investigation shows that, similar to GSK467, the planar bicyclic rings of DZNep and penciclovir wereCancers oriented 2020, 12, in the α-ketoglutarate binding pocket, and more significantly, the pyridine nitrogen10 of of15 DZNep and guanosine ketone of penciclovir were in contact with the metal center Mn2+ (Figure7). Theseobservations observations were weresimilar similar to the to binding the binding mode mode of GSK467 of GSK467 in inthe the JmjC JmjC domain domain of of human human KDM5B, wherewhere thethe pyridopyrimidinepyridopyrimidine nitrogennitrogen interactsinteracts withwith thethe metal metal center center (Figure (Figure7 )[7)20 [20]]. .

Figure 7. Comparison of binding modes of GSK467 (blue stick cartoon), penciclovir (yellow stick Figure 7. Comparison of binding modes of GSK467 (blue stick cartoon), penciclovir (yellow stick cartoon) and DZNep (green stick cartoon) in the JmjC domain of human KDM5B (pdb id: 5FUN). cartoon) and DZNep (green stick cartoon) in the JmjC domain of human KDM5B (pdb id: 5FUN). Mn2+ ion is shown as a green circle. Hydrogen atoms are removed to enhance clarity. Mn2+ ion is shown as a green circle. Hydrogen atoms are removed to enhance clarity. These results also show that small molecules, which possess planar bicyclic heterocyclic rings, exhibitThese favorable results binding also show toward that small the JmjC molecules domain, which of human possess KDM5B. planar Thisbicyclic is due heterocyclic to their ability rings, toexhibit undergo favorable interactions binding with toward the catalyticthe JmjC sitedomain metal of ion, human which KDM5B is an important. This is due criterion to their in ability KDM5 to inhibitionundergo interactions and is consistent with withthe catalytic previous site studies metal [20 ion,77, ,88which]. Our is molecular an important docking criterion investigations in KDM5 showinhibition that decitabine,and is consistent entecavir, with abacavir, previous penciclovir, studies [20,7 and7 DZNep,88]. Our have molecular the potential docking to beinvestigations repurposed asshow KDM5B that inhibitors decitabine, to treat entecavir, various abacavir, types of cancers. penciclovir, and DZNep have the potential to be repurposed as KDM5B inhibitors to treat various types of cancers. 5. Conclusions 5. Conclusions The leading role of KDM5B oncogene in tumorigenesis and cancer progression is well established in variousThe leading malignancies role andof KDM5B is therefore, oncogene considered in tumorigenesis as a potential therapeuticand cancer target. progre Althoughssion is many well smallestablished molecule in various inhibitors malignancies of KDM5B haveand is been therefore, identiﬁed considered so far, their as possiblea potential clinical therapeutic application target. in cancerAlthough treatment many is small a work molecule in progress. inhibitors This study of KDM5B provides have a rationale been identified on how repurposing so far, their antiviral possible clinical application in cancer treatment is a work in progress. This study provides a rationale on how repurposing antiviral drugs could contribute to the discovery and development of KDM5B inhibitors. We anticipate that these studies will stimulate further research in considering drug repurposing approaches to target KDM5B, and to discover novel therapies for various cancers.

Funding: This review received no external funding.

Acknowledgments: A.J. and L.T. are thankful for the Dr. TMA Pai PhD Scholarship from Manipal Academy of Higher Education, Manipal, India.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Sharma, S.; Kelly, T.K.; Jones, P.A. Epigenetics in cancer. Carcinogenesis 2010, 31, 27–36, doi:10.1093/carcin/bgp220. 2. Feinberg, A.P.; Tycko, B. The history of cancer epigenetics. Nat. Rev. Cancer 2004, 4, 143–153, doi:10.1038/nrc1279. 3. Dupont, C.; Armant, D.R.; Brenner, C.A. Epigenetics: Definition, mechanisms and clinical perspective. Semin. Reprod. Med. 2009, 27, 351–357, doi:10.1055/s-0029-1237423.

Cancers 2020, 12, 2121 11 of 16 drugs could contribute to the discovery and development of KDM5B inhibitors. We anticipate that these studies will stimulate further research in considering drug repurposing approaches to target KDM5B, and to discover novel therapies for various cancers.

Author Contributions: Conceptualization, M.R., P.P.N.R., and A.J.; writing—original draft preparation, A.J., P.P.N.R., and M.R.; writing—review and editing, A.J., M.R., P.P.N.R., G.R., G.G.S., J.K., N.A.N.K., G.S.R., M.M., and L.T. All authors have read and agreed to the published version of the manuscript. Funding: This review received no external funding. Acknowledgments: A.J. and L.T. are thankful for the TMA Pai PhD Scholarship from Manipal Academy of Higher Education, Manipal, India. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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